NaK-ATPase is the intrinsic enzyme of the plasma membrane that maintains the normal Na+ and K+ gradients across the surface membranes of most eucaryotic cells; and is the receptor for the positive inotropic actions of digitalis drugs in the myocardium. The long-term objectives of this project are to define the molecular mechanisms for the transmembrane ion movements through this enzyme, and to elucidate the mechanisms that control its function under physiological and pathophysiological conditions. The collaborative proposal is focused on the functional significance of the quaternary structure of the enzyme, and is based on the findings of the collaborating investigator suggesting that the subunit interactions of the duck nasal salt gland enzyme may differ significantly from those of the enzymes that have been used extensively by the Principal Investigator. The specific aims of the proposed comparative studies are: 1. To determine the stoichiometries of the active site and several ligand binding sites of the duck salt gland enzyme to see if this preparation exhibits half-site reactivity, and to compare these stoichiometries with the known stoichiometries of the kidney enzyme. 2. To examine the nature of the equilibrium binding plots for several ligands that interact with the duck salt gland enzyme to determine if these support the cooperative binding of ligands as suggested by the substrate-velocity curves of the enzyme. 3. To study ouabain binding to the duck salt gland enzyme, and to compare the effects of several physiological ligands of the enzyme on ouabain release from the salt gland and the kidney enzymes in order to characterize the multiplicity of the ligand binding sites in the two preparations. 4. To examine the subunit associations of the membrane-bound duck salt gland enzyme through chemical cross-linking studies. 5. To obtain active and stable detergent-solubilized preparations of the duck salt gland enzyme and the kidney enzyme, and to compare several properties of these preparations with those of the corresponding membrane-bound preparations.